Conjugation of polymers to biologically active moieties such as proteins, polypeptides or small molecules has become increasingly popular over the years as a means of increasing the effectiveness while often decreasing one or more negative aspects of such moieties. In particular, polymer conjugation using a polyalkylene oxide (PAO) or more specifically, polyethylene glycol (PEG), has been widely accepted for designing effective derivatives of semi-toxic or immunogenic drugs for therapeutic use.
U.S. Pat. Nos. 5,643,575; 5,919,455; 5,605,976 to name a few, describe non-antigenic polymers, their preparation and methods of conjugating them with biologically active moieties. The contents of each of the foregoing are incorporated herein by reference. Although these patents provide valuable methods for improving the use of biologically active moieties such as enzymes, proteins and other peptides, and polypeptides, there still exists a need for improved methods of conjugation.
For example, the aforementioned '575 patent describes methods of making branched PEG derivatives and protein conjugates made therewith. One method described therein involves using an excess of a trifunctional molecule such as, lysine ethyl ester to conjugate with activated mPEG derivatives such as succinimidyl carbonate (SC)-mPEG. While this method provides the desired activated branched polymer and resulting conjugate, it has been found that there are certain circumstances under which it would be desirable to more economically provide the desired conjugates in levels of higher purity. In the past, some have suggested using column chromatography to remove any unreacted starting materials or by-products. See, for example, U.S. Pat. No. 5,932,462. Such techniques are costly, are inconvenient for large scale manufacturing and can result in a significant loss of yield. It would be highly desirable to eliminate or inactivate the remaining starting materials and by-products which can compete with the formation of the desired branched polymer-therapeutic conjugates before the final product is made. This in turn would reverse the loss of yield. Thus, alternatives have been sought, especially in cases of commercial production and where it is critical to minimize loss of the biologically active moiety.
The present invention provides a new and improved process for the preparation of activated PEG linkers and their subsequent conjugation to biologically active moieties. The present invention also serves an unmet need to provide an economically efficient conjugation process having improved purity and yield over the prior art.